Overview

The CRISPR-Cas9 system is able to achieve highly specific and precise targeting for the genetic manipulation of cells and cell lines, transforming the field of genome engineering and presenting a wealth of new possibilities for R&D.

Why choose DefiniGEN?

DefiniGEN have optimized conditions for CRISPR Cas-9 genome editing on multiple patient-derived iPSC’s, and we have built up considerable experience in using the system for genomic manipulation in a range of other cell lines.

We can offer our clients an integrated end-to-end package including sgDNA design, transfection, optimisation, and clone picking/sequencing to generate a library of gene edits for client selection. From a basic frameshift knock-out mutation to complex knock-in genetic changes, our dedicated scientific team will work collaboratively with you to design the right tools to accelerate your research programs.

Workflow

We have developed a complete workflow for the knockout of an endogenous gene. Our workflow consists of footprint-free CRISPR/Cas9-mediated editing, single-cell cloning of the edited population, and characterization of expanded clonal cell lines to identify positive clones. The edited iPSC cells maintain pluripotency and have normal, stable karyotypes, which are important for testing downstream applications such as directed differentiation.

Disease Modelling

Our CRISPR services gives us the ability to repair causative mutations to patient-derived lines, or introduce alleles into a healthy parental line. The differentiated disease model human cells are a powerful tool to support preclinical research.

Monogenic diseases we can model

Monogenic Diseases

Gaucher disease

Acute intermittent porphyria

Genetic cholestasis (PFIC, TGP2, and Alagille syndrome)

Familial hypercholesterolemia

Wilson’s disease

Organic acidurias (except MSUD)

Hereditary hemochromatosis

Cystic fibrosis

Tyrosinemia type 1

Erythropoietic protoporphyria

Alpha-1 antitrypsin deficiency

MCAD deficiency

Arginosuccinic aciduria (ASL)

D-bifunctional protein deficiency

Glycogen storage disease (GSD) type 1

Galactosemia Type 1

Urea cycle disorders (except ASL)

Citrullinemia

Crigler-Najjar syndrome

Familial amyloid polyneuropathy

Primary hyperoxaluria type 1

Atypical haemolytic uremic syndrome 1

Maple syrup urine disease (MSUD)

When modelling any pathology using an iPSC differentiation platform, it is vital to confirm that the metabolic pathways implicated are expressed and active in the wild-type cells. DefiniGEN's hepatocytes are mature, predictive and display all relevant biological pathways pertinent for our disease models, as these data show below.

Wilson's Disease

The ATP7B gene is fundamental in hepatocytes for enabling the cells to effectively metabolise copper, and mutations in this gene can lead to Wilson’s disease. The ATP7B gene is shown here by qPCR to be expressed at comparable levels to human primary cells and the pathway is active in DefiniGEN’s differentiated hepatocytes, which make a good isogenic control for our disease model cells.

Gaucher's Disease

Gaucher’s disease is caused by the defective activity of the lysosomal hydrolase glucocerebrosidase, which is encoded by the GBA gene. The GBA pathway is present in DefiniGEN’s differentiated hepatocytes, shown here by qPCR, which provide a good isogenic control to our disease model cells.

Crigler-Najjar Syndrome

Crigler-Najjar syndrome is a rare monogenic disorder which is caused by a mutation in the UGT1A1 gene. The UGT1A1 enzyme is required for the conjugation and excretion of bilirubin from the body. The UGT1A1 pathway is shown here by qPCR to be expressed sufficiently in DefiniGEN’s wild type differentiated hepatocytes for them to be used as isogenic controls to our disease model cells.

Familial Transthyretin Amyloidosis

Familial Transthyretin Amyloidosis is a rare inherited condition characterized by abnormal build up of a protein called amyloid in the body. DefiniGEN hepatocytes present a good model system that mimic the disease phenotype and allow researchers to understand important disease-related mechanisms. This graph shows expression of the TTR pathway in our control wild type hepatocytes as comparable levels to human primary cells.

Alagille Syndrome

Alagille Syndrome is caused by mutations in the JAG1 and NOTCH2 genes. The NOTCH2 and JAG1 gene expression is shown here to be at comparable levels to human primary cells in DefiniGEN’s differentiated hepatocytes, and therefore provide a good isogenic control to our disease model cells.

Hereditary Haemochromatosis

The HFE gene is central in hepatocytes for enabling the cells to effectively metabolize iron, and mutations in this gene can lead to Haemochromatosis. The HFE pathway is shown here by qPCR to be expressed at sufficient levels in DefiniGEN’s differentiated hepatocytes, to provide a good isogenic control to our disease model cells.